Abstract A092: The SPAN Score as the Primary Outcome for Multicenter Preclinical Randomized Controlled Stroke Trials

Background: Selecting a robust, generalizable primary outcome is critical for multicenter preclinical stroke trials. The Stroke Preclinical Assessment Network is a randomized, placebo-controlled, blinded, multi-laboratory trial using a multi-arm, multi-stage design to evaluate interventions with high translational potential. During a SPAN 2.0 pilot study, we developed the SPAN score to capture functional deficits after middle cerebral artery occlusion (MCAO). Reliability and validity analyses led to a 6-item weighted version. This study evaluated (1) the performance of the weighted score in the SPAN 2.0 pilot dataset and (2) its external validation in independent SPAN 2.1 and 2.2 cohorts to determine suitability as the standardized primary outcome for multicenter preclinical stroke research. Methods: Data from SPAN 2.0, 2.1, and 2.2 were analyzed. In the pilot, mice underwent 30- or 60-minute MCAO to produce mild to severe injuries. Outcomes included behavioral assessments, MRI, and survival. Probabilistic Index modeling estimated the probability that a randomly selected 30-minute outperformed a 60-minute MCAO animal (0.5 = no difference, >0.5 favors 30-minute). Kaplan–Meier curves and Cox models assessed mortality. Receiver Operating Characteristic (ROC) analysis tested the SPAN score’s ability to distinguish stroke from non-stroke and detect lesion volumes > 5 mm3 and > 10 mm3. Worst-score imputation was applied for deaths before data collection. Results: In SPAN 2.0, survival was higher after 30-minute MCAO (chi^2 = 4.17, p = 0.04). The weighted SPAN score was worse in the 60-minute group (p = 0.036). Differentiating measures included day-2 Bederson score, day-30 Novel Object Recognition, day-2 MRI infarct volume with midline shift, and day-30 CSF volume; several behavioral tests did not significantly separate groups. In SPAN 2.1 and 2.2, with worst-score imputation, the SPAN score showed greater impairment in aged vs. diabetes-induced (p = 0.022) and young healthy mice (p < 0.001). ROC confirmed discriminatory ability: infarct vs. none, AUC = 0.747 (p < 0.001); infarct > 10 mm3 vs. none, AUC = 0.774 (p < 0.001). Conclusion: The 6-item weighted SPAN score was the most suitable primary outcome in the SPAN 2.0 pilot. External validation in SPAN 2.1 and 2.2 confirmed its consistent ability to differentiate stroke severity across models and sites, supporting its adoption as the standardized primary outcome for future multicenter preclinical stroke trials.